Axon Guidance Proteins in Mammary Gland Development

Institution: University of California, Santa Cruz
Investigator(s): Lindsay Hinck, Ph.D. -
Award Cycle: 2004 (Cycle 10) Grant #: 10PB-0188 Award: $449,228
Award Type: Request for Applications
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2004)
Many studies have shown that one of the first changes observed in the development of solid malignant tumors, such as breast tumors, is loss of normal tissue morphology. The Slits are a family of secreted proteins that are candidates for mediating interactions betweens cells and their environment. They were originally discovered in the nervous system where they repel and attract axons, and promote their branching. Receptors for Slits, called Robos, have also been discovered in the nervous system and appear to mediate the many, diverse functions of Slits. Members of both the Slit and Robo families are expressed in other developing organs, but their function in these tissues is unknown. Our preliminary data shows that Slit2, Slit3, Robo1 and Rig1 (Robo3) are expressed in the mammary gland and there is growing evidence that these genes function as mammary tumor suppressors. Given so little information about the normal role of Slits outside the nervous system, we do not understand how their loss of expression during cancer progression affects their function. The aim of this grant is to elucidate the biology of Slits and Robos in the developing mammary gland by analyzing their loss-of-function phenotypes. With these studies we will establish the foundation for understanding the role these genes play as tumor suppressors in breast.

Symposium Abstract (2005)
Phyllis Strickland1, Grace C. Shin1, Adam Boyette1, Andrew Plump2, Marc Tessier-Lavigne3 and Lindsay Hinck1*

1Department of Molecular, Cell and Developmental Biology University of California, Santa Cruz Santa Cruz, CA 95064, 2Merck Research Laboratories, 126 Each Lincoln Avenue, Rahway, New Jersey 07065, 3Genentech, Incorporated, 1 DNA Way, South San Francisco, CA 94080

SLITs, secreted guidance cues, and their receptors, ROBOs, were originally identified based on their role in organizing neural connections during nervous system development. They are also present in other developing organs, including breast. Many genes that contribute to normal development are present at inappropriately levels during tumor progression. Indeed Slit and Robo genes are frequently inactivated in breast cancer patients and recent evidence from human breast tumors and tumor cell lines suggests that Slits normally function as tumor suppressors (Dallol et al. Cancer Research 62, 2002). The aim of our studies is to establish a foundation for understanding how these genes function as tumor suppressors in breast by analyzing the consequences of their loss-of-function during normal mouse mammary gland development.

We demonstrate that SLIT2 signals through ROBO1 to generate organized cell-cell contacts during mammary ductal development. ROBO1 expression is restricted to cap/myoepithelial cells, whereas SLIT2 is broadly distributed. Loss-of-function mutations in either Slit2 or Robo1 yield similar defects, characterized by disorganized end buds that are reminiscent of those present in Netrin-1(Ntn1) -/- glands, suggesting that SLIT2 and NTN1 function in concert during mammary development. Glands harboring double homozygous mutations in Slit2 and Ntn1 exhibit enhanced defects, displaying significant ductal dysplasia characterized by loss of adhesion between myoepithelial and luminal cell layers and ductal occlusion. To investigate the mechanism underlying this disorganization, Slit2-/-; Ntn1-/- cells were incubated in rotary cultures. We observe that Slit2-/-; Ntn1-/- cells, in contrast to normal cells, do not form bi-layered organoids, a defect that is rescued by the addition of purified SLIT2. NTN1 has no effect alone, but synergistically enhances this rescue.

Thus, our data identify two adhesive guidance systems, SLIT2/ROBO1 and NTN1/NEO1 that act in parallel to generate cell boundaries during mammary gland development.

Symposium Abstract (2007)
Development of many organs, including the mammary gland, involves dramatic changes in shape and form as tissues are molded into three-dimensional structures. The mammary gland is a tree-like structure composed of bi-layered ducts, comprising an outer layer of cap/myoepithelial cells and an inner layer of luminal epithelial cells surrounding a central lumen. During development, the enlarged termini of ducts, termed end buds, establish the primary ductal architecture and drive the prodigious growth of the gland that establishes the mammary tree.

My laboratory is interested in understanding mechanisms that regulate ductal architecture and we have identified two signaling systems responsible for adhesive interactions between the bi-layered sheets of mammary epithelium. SLITs (Slit), like NETRINs (Ntn), were originally characterized as guidance cues that direct neurons and their axons to targets during neural development. In mammary gland, SLIT2 and NTN1 are broadly distributed throughout the epithelial compartment, whereas their receptors, ROBO1 (Robo1) and NEOGENIN1(Neo1), are restricted to the outer myoepithelial cell layer. Loss-of-function mutations in any one of these genes results in adhesive defects that are confined to the end bud structure. Ductal defects, in which the luminal epithelial cell layer peels away from the myoepithelial cell layer, are only revealed when both Slit2 and Ntn1 are deleted. These and other studies have led us to propose a model in which the two cues act in parallel through their respective receptors to mediate adhesive interactions between distinct epithelial cell types. We suggest that this type of short-range adhesion maintains tissue structure, while allowing cell movement and re-organization during periods of rapid tissue growth and remodeling.

Our studies on normal mammary development have also revealed a potential role for SLITs as tumor suppressors. We found that loss of both Slit2 and Slit3 in the mammary epithelium leads to increased proliferation and ductal lesions. To investigate, we identified candidates whose misexpression may contribute to the invasive phenotype. We discovered a dramatic upregulation of the chemokine receptor CXCR4 in Slit2-/-;Slit3-/- epithelium, and a concomitant upregulation of its ligand SDF1 (CXCL12) in the surrounding stroma. The SDF1/CXCR4 signaling pathway is emerging as a key player in human breast cancer with roles in both regulating the growth of primary tumors and in directing the migration of metastasizing cells. With our on-going studies, we are investigating how loss of Slits and their Robo receptor leads to changes in Sdf1/Cxcr4 expression and the consequences of this in breast cancer progression.

SLITs suppress tumor growth and microenvironment in vivo by silencing Sdf1/Cxcr4 within breast epithelium
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Marlow, R., Strickland, P., Lee, J., Wu, X., PeBenito, M., Binnewies, M. Le, E., et al.
Yr: 2008 Vol: 68 Nbr: Abs: Pg:7819-7827